Type II restriction endonucleases (REs) are highly sequence-specific compared with otherclasses of nucleases. PD-(D/E)XK nucleases, initially represented by only type II REs, now comprisea large and extremely diverse superfamily of proteins and, although sharing a structurally conservedcore, typically display little or no detectable sequence similarity except for the active site motifs.Sequence similarity can only be observed in methylases and few isoschizomers. As a consequence,REs are classified according to combinations of functional properties rather than on the basis ofgenetic

relatedness. New alignment matrices and classification systems based on structural coreconnectivity and cleavage mechanisms have been developed to characterize new REs and

relatedproteins. REs recognizing more than 300 distinct specificities have been identified in REdatabase

(REBASE: http://rebase.neb.com/cgi-bin/statlist) but still the need for newer specificities is increasingdue to the advancement in molecular biology and applications. The enzymes have undergoneconstant evolution through structural changes in protein scaffolds which include random mutations,homologous recombinations, insertions, and deletions of coding DNA sequences but rationalmutagenesis or directed evolution delivers protein variants with new functions in accordance withdefined biochemical or environmental pressures. Redesigning through random mutation, addition ordeletion of amino acids, methylation-based selection, synthetic molecules, combining recognition andcleavage domains from different enzymes, or combination with domains of additional functionschange the cleavage specificity or substrate preference and stability. There is a growing number ofpatents awarded for the creation of engineered REs with new and enhanced properties.